This invention relates generally to a novel method of direct serum assay of steroid hormones of the type conducted without the need to extract the hormones from the serum prior to assay. More particularly, this invention concerns reducing or eliminating the binding protein competition from interacting with steroid hormones prior to the stage in the assay when the bound or unbound antigen (tagged and untagged) is separated for counting, by adding an artificial compound having a greater or equal affinity for the binding protein than do the steroid hormones, and which is non-immuno-reactive with the steroid hormones. Because the compound is artificial, it is non-contaminating.
In the past, it has been common practice to assay for a selected antigen, e.g., one of the steroid hormones, by employing a model antigen and an antibody which is specific to the antigen it is desired to assay, which model antigen is tagged in some way, e.g. with a radio-nuclide or an enzyme. In such an assay, e.g., a radioimmuno-assay, a radioactively tagged antigen, and the specific antibody are added to the serum. The added antibody is made specific by, e.g., in the case of the human steroid hormone estradiol, estradiol coupled to a protein may be added in vivo to an animal, e.g., a rabbit. The rabbit's system will produce an antibody to the estradiol. The model, i.e., tagged, antigen is added in a quantity sufficient to exceed the number of binding sites available in the amount of added antibody. Typically, the tagged antigen will be added to exceed the antibody by at least a factor of two. Thus, typically, after addition, the serum will contain: EQU 2Ag*+Ab
where the * indicates the model or tagged antigen is modified in some way, as by tagging with a radionuclide or an enzyme, which will enable quantitation in the assay.
Once the tagged antigen complexes with the available specific antibody the serum will contain: EQU (Ag*Ab)+Ag*
which is typically referred to as bound [(Ag*Ab)] and unbound [Ag*]. To conduct the assay it then is only necessary to separate the bound from the unbound fractions and compare the amount of radioactivity (or other tagging mechanism), in either of the separated portions of the serum containing the bound or the unbound fraction, with the total amount of radioactivity (or other tagging mechanism) initially added, then by use of a standard curve as is shown in FIG. 1 the amount of the untagged hormone or antigen which it is desired to assay can be determined.
The curve shown as a solid line in FIG. 1 is the standard curve for determining the amount of target antigen present in a sample being assayed by counting the amount of tagged antigen bound to the specific antibody. As can be seen, the larger the amount of tagged bound antigen in the bound fraction, which includes both tagged (model) and untagged (target) antigen bound to the specific antibody (in the case of a radioactive tagging illustrated in FIG. 1), the higher the percentage counts per minutes, compared to the total counts per minute of the added tagged antigen, and the lower the concentration of the target hormone being assayed. This is due to the fact that both the added antibody which is necessary to bind the antigen (tagged or untagged) in a given sample of serum and the amount of tagged antigen added are constants. The standard curve is developed by decreasing the ratio of the tagged to the untagged antigen, specific to the antibody in standardized sample runs with known amounts of untagged antigen. In the assay itself, tagged antigen and antibody are added to the serum which already contains the natural target antigen. Because both tagged and untagged antigen compete for sites on the antibody (which is in a known constant amount and specific to the added tagged antigen and untagged antigen present in the serum sample) to complex with that antibody, a higher amount of tagged antigen measured in the separated bound Ag*Ab complex indicates a smaller concentration of untagged antigen for which the assay is conducted.
Correspondingly, the percentage of the tagged antigen remaining in the separated unbound portion of the serum increases as the amount of available untagged antigen, for which the assay is being conducted, increases in the serum sample.
As is well known in the art, the bound fraction can be separated from the unbound by adding to the serum a further antibody from a different species which is specific to the antibody so that the antigen antibody complex (tagged and untagged), i.e., the bound antigens, in the serum and the resultant double antibody complex will precipitate out, leaving the unbound tagged and untagged antigen in the supernatant of the serum sample. For example, if the antibody, an immunoglobulin, is produced in rabbits, the rabbit serum selected to be specific to the antigen to be assayed, an antibody from, e.g., a donkey serum, specific to the rabbit immunoglobulin will complex with the rabbit Ag*Ab tagged complex and the untagged AbAg complex in the serum of the assay. The sheer size of this new complex will cause it to precipitate out of the serum leaving the unbound Ag* and the unbound untagged Ag in the supernatant of the serum.
Also, as is well known in the art, the unbound Ag* and untagged Ag can be separated by adding a fine charcoal suspension which will absorb the Ag* and untagged Ag by surface adhesion to the charcoal. Centrifuging will then separate the absorbed unbound Ag* and untagged Ag from the serum leaving the bound Ag*Ab complex and the bound untagged AgAb complex in the supernatant.
The problem known in the art in assaying sex steroid hormones from, e.g., human serum samples, is that the sex steroid hormones occur in such minute quantities. Such quantities are typically on the order of from about 5 to about 700 picogram/ml for, e.g., estrogens, depending on the age and other biological factors pertaining to the patient from whom the serum is drawn and the hormone cannot be quantitively extracted with organic solvents. This results in the rigid requirement that the amount of hormone extracted by the state of the art methods be known, resulting in extra steps to ensure that the results are accurate. Presently known methods utilize the addition of a very minute known amount of tagged antigen (as a tracer) to the serum prior to extraction followed by counting the extract to determine the percent of antigen extracted. For direct serum assay, the low recovery results in the placing of the bound and unbound fractions on the flat portions of the standard curve shown in FIG. 1. Thus, the ability to determine the amount of the sex steroid hormone in a direct assay in the serum, based upon either a variation in the amount of tagged antigen remaining in the unbound fraction or in the amount of tagged antigen complexing into the bound fraction, is not readily possible. What is possible in the prior art methods is to measure the apparent rather than the actual amount of steroid hormone by shifting the standard curve. However, since the amount of steroid hormone unbound by SHBG in any given patient varies at any given time from approximately 2% to approximately 30%, callibration of the shifted standard curve is not possible. The problem of being on the flat portion of the standard curve could be circumvented by extracting about an order of magnitude larger quantity of serum (i.e., blood) from the patient, to increase the available quantities of steroid hormone sufficiently to shift the standard curve, but when numerous hormones are to be tested from one patient, this becomes an impractical amount of serum extraction. However, apparent and not actual steroid hormone content is being measured due to the inability to know the percent bound by the binding protein.
The factor which accounts for this set of requirements is the presence in the serum of the binding protein, referred to in the art as either SHBG or SSBG or TeBG, and also as simply BP. SHBG has a great affinity for the steroid hormones which will compete with the antibody. This binding protein is also responsible for the incomplete extraction. In human serum, the, e.g., sex steroid hormones exist in the free, i.e., unbound to SHBG, state in typically from 2-30% of the total available sex steroid hormone. Since the binding protein has about the same affinity for sex steroid hormones as the antibody, little displacement of the bound sex steroid hormones to the antibody from the binding protein occurs when the antibody is added to the serum. Therefore, typically only about 2-30% of the sex steroid hormone, i.e., that amount unbound to the binding protein in the sample serum, is available for competing with the added tagged antigen to complex with the added antibody. This is the basis for the decrease of the available sex steroid hormone for competing with the added tagged, e.g., with radio-nuclide, antigen, hindering the direct radioimmunoassay of sex steroid hormones.
Thus for example, with estradiol in human serum EQU SHBG+E.sub.2 .revreaction.(SHBG-E.sub.2)
The association constant for this SHBG-E.sub.2 complex is 10.sup.9 liters/moles with the corresponding dissociation constant being 10.sup.-9 (liters/moles.sup.-1). The dissociation constant for the free antibody is approximately 3.times.10.sup.-10 (liters/moles.sup.-1). So long as this is within an order of magnitude of that for SHBG, the SHBG will very effectively compete with the added tagged antigen for sites to complex with estradiol.
In the past it has been known to extract the steroid hormones in the serum sample by using an appropriate organic solvent. This process takes time and several steps to complete, since extraction of the hormone is incomplete, both of which make such a procedure unattractive to a commercial laboratory in the business of analyzing serum samples for the presence of, among other things, steroid hormones.
It has also been known to add dihydrotestosterone (DHT) to free more of the, e.g., sex steroid hormones from the binding protein due to the greater affinity of SHBG for DHT than for the other sex steroid hormones. This addition of DHT places a greater amount of the other sex steroid hormones in the free state in the serum. Assay then can be accomplished of this greater amount of other free sex steroid hormones. A problem which occurs in using this technique is that DHT also occurs in sample serums in amounts comparable to the other sex steroid hormones and is often desired to be measured. Using DHT in the commercial laboratory setting to free the sex steroid hormones from the binding protein for assay thereof, creates the significant potential for contaminating the laboratory, its technicians or equipment, with DHT, thus interfering with other measurements being conducted of DHT. Because DHT occurs in such small quantities in serum samples it would have to be added in such relatively large quantities to displace the, e.g., sex steroid hormones from the SHBG, the potential for significant DHT contamination of the laboratory exists. It is further known in the art as disclosed by Green, Marshall, Pensky and Stanbury, "Thyroxin Binding Globulin: Characterization of the Binding Site with a Fluorescent Die as a Probe," Science, Vol. 175, at 1378 (1972), that ANS (8-anolino 1-naphthalenesulfonic acid) can be used to displace the thyroid hormone from thyroxine binding protein (TBG--thyroxine binding globulin) for direct assay purposes. ANS is an allostere of thyroid hormone for TBG. Also known, as described in Edward U.S. Pat. No. 4,208,400, is the incorporation of progesterone in an incubated assay mixture containing unconjugated oestriol. Here too, a significant potential for contamination exists, interfering with other assays, for example, the assay of progesterone itself.
While such prior art methods have exhibited at best a degree of utility, room for significant improvement in the methods of assay of steroid hormones of the prior art remains. The need clearly exists for a direct serum assay of steroid hormones without the need to add DHT to the serum and which can be conducted without the fear of contamination of other test procedures.
The problems enumerated in the foregoing are not intended to be exhaustive, but rather are among many which tend to impair the effectiveness of previously known methods of assay of steroid hormones. Other noteworthy problems may also exist; however, those presented above should be sufficient to demonstrate that methods of assay of steroid hormones appearing in the prior art have not been altogether satisfactory.